Virus-Viroid Interactions: Citrus Tristeza Virus Enhances the Accumulation of Citrus Dwarfing Viroid in Mexican Lime via Virus-Encoded Silencing Suppressors

Abstract

An assay to identify interactions between Citrus dwarfing viroid (CDVd) and Citrus tristeza virus (CTV) showed that viroid titer was enhanced by the coinfecting CTV in Mexican lime but not in etrog citron. Since CTV encodes three RNA silencing suppressors (RSSs), p23, p20 and p25, an assay using transgenic Mexican limes expressing each RSS revealed that p23 and, to a lesser extent, p25 recapitulated the effect observed with coinfections of CTV and CDVd. V iroids are infectious, unencapsidated, single-stranded, and highly structured noncoding circular RNAs of 250 to 400 nucleotides (nt). Despite their simplicity, viroids interact with cell factors, which redirect them to replicate and traffic throughout the plant while avoiding the RNA silencing machinery they trigger in their hosts (1).Indexing tests have shown that viroids are widespread in commercial citrus orchards, wherein trees may be coinfected with other graft-transmissible pathogens. Data regarding virus-viroid interactions are very scarce, but the information available indicates that such interactions may alter symptom expression (2, 3). Since coinfection of citrus species with Citrus tristeza virus (CTV) and viroids is not rare, assays were conducted to establish whether interactions between these two types of pathogens actually exist.The assays were performed by comparing single and double infections of the two pathogens in etrog citron (Citrus medica L.) (viroid bioindicator) and Mexican lime (Citrus aurantifolia [Christm.] Swingle) (CTV bioindicator). Citrus dwarfing viroid (CDVd) (GenBank accession number EU934004), inducing moderate but specific symptoms in etrog citron (4), and CTV (T-305 strain), inducing symptoms in Mexican lime (5), were chosen for this study. To synchronize infection, Volkamer lemon (Citrus volkameriana Ten. & Pasq.) seedlings were graft inoculated with CDVd and/or CTV before being used as rootstocks. Etrog citron and Mexican lime were graft propagated to produce groups of five plants for each of the following treatments: (i) plants singly infected with CDVd, (ii) plants singly infected with CTV, (iii) plants coinfected with CTV and CDVd, and (iv) noninfected controls. CTV and CDVd infection was assessed by immunoprinting enzyme-linked immunosorbent assay (ELISA) (6) and dot blot hybridization (7), respectively.After an incubation period of 4 months at 25°C, plant height was measured (Fig. 1) and data were subjected to multifactor analysis of variance (ANOVA). The results indicated that etrog citron singly infected with CTV (P value, 0.00001) or CDVd (P value, 0.05) was stunted, as was Mexican lime singly infected with CTV (P value, 0.0013) but not that with CDVd (P value, 0.98). Height reduction in coinfected plants resulted from the added effect of each pathogen, indicating no interaction between CDVd and CTV in either host (interaction P value for etrog citron, 0.29; interaction P value for Mexican lime, 0.44). Similarly, symptom expression in stems and leaves resulted from the added effect of each pathogen, revealing agai...

Specific Argonautes Selectively Bind Small RNAs Derived from Potato Spindle Tuber Viroid and Attenuate Viroid Accumulation In Vivo

“…From an alternative perspective, the secondary structure of viroids could have emerged as a compromise between resistance to DCL and to RISC, which act preferentially against RNAs with compact and relaxed conformations, respectively (48). Indeed, data obtained in other experimental contexts indicate that viroids are RISC sensitive (46,48,49,52), and recent results show that RISC promotes cleavage of viral RNAs with a packed secondary structure-resembling that of viroids-by targeting bulged regions within the structure (72). However, the evidence that one or more AGOs are loaded with vd-sRNAs and function in antiviroid RISC is circumstantial, with no data providing direct support for this view.…”

“…Fourth, two vd-sRNAs containing the albinism determinant of a chloroplast-replicating viroid guide cleavage of the host mRNA coding for the chloroplastic heat shock protein 90 (cHSP90) as predicted by RNA silencing, thus providing a feasible mechanism of pathogenesis (51). Fifth, the titer of a viroid is enhanced in coinfections with a virus, with this effect occurring via the expression of virus-encoded silencing suppressors (52). Last but not least, early pioneering research discovered that PSTVdcDNA introduced into the tobacco genome via Agrobacterium tumefaciens became methylated only following viroid RNA-RNA replication, thus revealing an RNA-directed and sequence-specific mechanism for de novo methylation of genomic sequences in plants (53); this mechanism of transcriptional silencing is now known to be mediated by specific DCL-dependent siRNAs and AGOs.…”

A mutual titer-enhancing relationship and similar localization patterns between Citrus exocortis viroid and Hop stunt viroid co-infecting two citrus cultivars

“…We were unable to discern the mechanism of interaction between the two viroids at macroscopic or microscopic levels. A previous study, in which titers of Citrus dwarfing viroid (CDVd) were enhanced by the co-infecting Citrus tristeza virus (CTV) in Mexican lime, provides a possible clue: the CTV RNA silencing suppressor p23 aided CDVd invasion and replication [ 7 ]. Our results suggest that temperature might be another factor affecting the interaction between the two viroids (Table 2 ).…”

“…A possible mechanism for virus/viroid interaction was uncovered in a study using viral-encoded silencing suppressors, where a titer of Citrus dwarfing viroid (CDVd) was enhanced by Citrus tristeza virus (CTV) in Mexican lime but not in Etrog citron. Interactions between these pathogens differed among host plant cultivars, indicating that such interactions are likely dictated by the host [ 6 , 7 ]. In viroid/viroid interactions, multiple viroids in various citrus hosts show complicated antagonistic or synergistic relationships that lead to different symptoms, canopy volumes, fruit yields and commercial performance.…”

Engineering Viroid Resistance

“…This is reminiscent of a tobacco in vitro system, in which overexpression of AGOs was shown to be required to achieve efficient RNA silencing [ 47 ]. Finally, (5) co-infection experiments of Citrus dwarfing viroid (CDVd) and Citrus tristeza virus (CTV) led to elevated viroid titre due to CTV’s silencing suppressors [ 48 ], suggesting that viroids could not fully evade RNA silencing under native conditions. Viroids do not encode any suppressors of silencing and replicate in subcellular compartments (nucleus or chloroplast) where vd-siRNAs-mediated PTGS seems to be absent [ 29 ].…”